Dysferlin: Approaches to Domain Analysis and Gene Therapy

膜修复是大型、不可替代或经常受损的细胞的基本生存过程 比如肌肉细胞。我们已经报道了三吉的骨骼肌蛋白异铁蛋白缺陷。 肌病(MM)和肢体带状2B型肌营养不良症(LGMD2B)以及异铁蛋白加速肌肉 膜修复通过钙触发的机制，调用与膜联蛋白的结合和形成 胞内囊泡聚集体。因此，MM和LGMD2B似乎很可能是异常的结果 骨骼肌膜修复。我们现在建议定义对此至关重要的deferlin结构域 修复过程并确定截短形式的“微型干扰素”是否能挽救MM/LGMD2B 表型。我们的研究有五个具体目标：(1)建立和鉴定过量表达的转基因小鼠。 表达全长和截短的干扰素。假设：全长或超正常水平的表达 截短的去铁蛋白可以挽救缺乏去铁蛋白模型中的营养不良表型。(2)描述 截短型去铁蛋白的生化性质。假设：全长和截短的生化分析 迪弗林蛋白将确定保留全长蛋白功能特性的结构域。(3) 微小脱铁蛋白对膜修复的影响。假说：迷你异铁蛋白保留 功能域将增强去铁蛋白缺乏的肌肉细胞的膜修复。(4)静脉分娩 候选AAV8包装的迷你异铁蛋白基因。假设：系统传递微小的异铁蛋白基因将 引起这些基因在肌肉中的传播，并加强膜修复。(5)分析斑马鱼 对于异铁蛋白的存在和当异铁蛋白同源物下调时的表型。 假设：功能障碍蛋白缺乏会在斑马鱼中产生一种肌病表型，这将是可能的 分析在斑马鱼中替换部分或全部形式的脱铁蛋白的影响。方法：我们将使用 各种技术来定义deferlin的功能结构域，然后将分析拯救 全长和截短异铁蛋白通过转基因进入肌肉的缺陷性表型 小鼠和系统注射AAV8。意义：这项研究将(1)加深我们对 骨骼肌肌膜修复的分子生物学；(2)提供对骨骼肌肌膜修复的深入了解 Deferlin的功能结构域；(3)确定开发微型deferlins用于拯救白血病的可行性。 缺铁素缺乏导致的肌肉表型；以及(4)开发关于以下方面的疗效的初步数据 系统性病毒基因治疗MM和LGMD2B。

Membrane repair is a fundamental cell survival process in large, irreplaceable, or frequently injured cell types such as muscle cells. We have reported that the skeletal muscle protein dysferlin is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and that dysferlin accelerates muscle membrane repair via a calcium-triggered mechanism that invokes binding to annexins and the formation of intracellar vesicular aggregates. It thus seems likely that MM and LGMD2B are a consequence of aberrant skeletal muscle membrane repair. We now propose to define domains of dysferlin that are critical for this repair process and determine whether truncated forms of "mini-dysferlin" can rescue the MM/LGMD2B phenotype. Our study has five Specific Aims: (1) Generate and characterize transgenic mice that over- express full-length and truncated dysferlin. Hypothesis: expression of supra-normal levels of full-length or truncated dysferlin can rescue the dystrophic phenotype in a dysferlin-deficient model. (2) Characterize the biochemical properties of truncated dysferlin. Hypothesis: A biochemical analysis of full-length and truncated dysferlin proteins will identify domains retaining functional properties of the full-length protein. (3) Characterize membrane repair by mini-dysferlin proteins. Hypothesis: mini-dysferlin proteins retaining functional domains will augment membrane repair in dysferlin-deficient muscle cells. (4) Intravenously deliver candidate AAV8-packaged mini-dysferlin genes. Hypothesis: systemic delivery of mini-dysferlin genes will give rise to dissemination of these genes in muscle and enhanced membrane repair. (5) Analyze zebrafish for the presence of dysferlin and for phenotypes when dysferlin homologues are down-regulated. Hypothesis: Dysferlin-deficiency will produce a myopathic phenotype in zebrafish and it will be possible to analyze the impact of replacement of partial or full forms of dysferlin in the zebrafish. Methods: We will use a variety of techniques to define functional domains of dysferlin and will then analyze the rescue of the dysferlin-deficient phenotypes by full length and truncated dysferlin delivered to muscle in vivo via transgenic mice and systemically administered AAV8. Significance: This study willl (1) enhance our understanding of the molecular biology of sarcolemmal membrane repair in skeletal muscle; (2) provide insight into the functional domains of dysferlin; (3) determine the feasibility of developing mini-dysferlins for rescue of muscle phenotypes resulting from dysferlin deficiency; and (4) develop pilot data on the efficacy of systemically administered viral gene therapy for MM and LGMD2B.